Notch proteins are the receptors in a highly conserved signal transduction system used to communicate information between cells that contact each other. The overarching goal of this Project is to elucidate the mechanism by which canonical ligands expressed on signal-sending cells activate Notch receptors on signal-receiving cells. Specifically, we propose two complementary sets of studies that will decipher two of the critical events that are required for ligand-induced Notch signaling: Aim 1. To determine how Mind bomb induces ligand-dependent Notch signaling We will exploit the modular nature of mib and our expertise in structural and biochemical methods to determine the molecular logic underlying mind bomb function. Our top priorities will be i) to determine the structural basis for ligand-tail binding, and ii) to determine how the different Mib domains cooperate to transfer ubiquitin onto these tails. Aim 2. To determine how ligand stimulation induces metalloprotease cleavage of Notch receptors One leading model for ligand-dependent activation of Notch posits that the endocytosis of bound ligand exerts a mechanical force on the receptor, releasing autoinhibitory interactions that protect the metalloprotease cleavage site. These studies will combine powerful single-molecule approaches and cellbased assays to evaluate the feasibility of the mechanical force model of Notch signal induction. Distinguishing between a mechanotransduction model and alternatives, such as allosteric conformational switch models, will substantially advance our understanding of the key events required for conveying Notch signals between adjacent cells, and will have important implications for efforts to target ligand-dependent Notch signaling. Moreover, the methods developed for probing the effect of mechanical force in Notch signal transduction will have general application in many other fields where the role of force in biology is under investigation. Together, pursuit of these aims will substantially advance our understanding of the key steps responsible for conveying Notch signals between adjacent cells. In addition, they will identify new potential targets for interventions designed to interfere with oncogenic Notch signaling in a wide spectrum of human cancers.